Biomedical subjects
Publications and source records attributed to E Drouet.
Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.
Epstein-Barr virus (EBV), a member of the gamma-herpesvirus family, is involved in the development of several diseases, and the infection is believed to persist for life in latent form. Ionizing radiation at clinically relevant doses may increase the amount of virus reactivation in B cells, and the combination of radiation with stress could amplify EBV reactivation. In vitro experiments were performed on several cell lines, including EBV-positive Burkitt lymphoma cells. The presence of the immediate-early protein ZEBRA, which is a hallmark of EBV reactivation, was evaluated using flow cytometry, which enabled us to measure the percentage of ZEBRA-positive cells. The process was studied previously in the EBV-positive Burkitt lymphoma cell line B95-8. Forty-eight hours after irradiation alone, 13.6 and 19.9% ZEBRA-positive cells were observed at 2 and 4 Gy, respectively, compared to the basal level of 1.85%. Thus irradiation induces EBV reactivation. The addition of a glucocorticoid (the final effector of the stress response) had no effect on EBV reactivation in our model. However, the combination of radiation and treatment with a glucocorticoid (dexamethasone) increased the expression of ZEBRA in B95-8 cells (15.8 and 28.75% of the cells was positive at 24 and 48 h after gamma irradiation, respectively). Thus the combination of gamma radiation and a glucocorticoid may play an important role in EBV reactivation.
SPARSE DATA: The Gulf war syndrome remains a little know entity since its first appearance 10 years ago. The objective of our work was to synthesize the data published on the subject in the scientific literature. We analysed the results of American and English epidemiological surveys, from which it was difficult to distinguish the existence of a univocal syndrome. IMPRECISE DEFINITION: It is difficult to give a clear clinical definition of the syndrome, the signs of which fluctuate depending on the studies. Chronic fatigue is frequently associated with the Gulf war syndrome, although some studies have described electrophysiological neurological lesions. NUMEROUS HYPOTHESES: The role of stress, vaccinations and their adjuvants, exposition to neurotoxic substances and weak uranium have been incriminated. We propose that multiple factors be integrated in the research for the genesis of this atypic syndrome.
Several recent epidemiological studies have shown that vaccinations against biological warfare using pertussis as an adjuvant were associated with the Gulf war syndrome. If such epidemiological findings are confirmed, we propose that the use of pertussis as an adjuvant could trigger neurodegeneration through induction of interleukin-1beta secretion in the brain. In turn, neuronal lesions may be sustained by stress or neurotoxic chemical combinations. Particular susceptibility for IL-1beta secretion and potential distant neuronal damage could provide an explanation for the diversity of the symptoms observed on veterans.
Adenoviruses of serotype Ad3 (subgenus B) use a still-unknown host cell receptor for viral attachment, whereas viruses from all other known subgenera use the coxsackie and adenovirus receptor (CAR). The receptor binding domain (head) of the Ad3 fiber protein has been expressed in Escherichia coli inclusion bodies. After denaturation and renaturation using a rapid dilution method, crystals of trimeric head were obtained. The 1.6 A resolution X-ray structure shows a strict conservation of the beta-sheet scaffold of the protein very similar to the head structures of the CAR-binding serotypes Ad2, Ad5, and Ad12. The conformation of the loops is different, with the exception of the AB loop, which forms the center of the interface in the Ad12-CAR complex structure. The structure explains why a mutation in Ad5 of one residue in the AB loop to glutamic acid, as in Ad3, abrogates binding to CAR. It is possible that the Ad3 receptor binding site is nevertheless situated similar to the CAR binding site, although it cannot be excluded that other regions of the relatively hydrophobic head surface may be used.
Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells.
The purpose of the present study was to assess the viral diversity of hepatitis C virus (HCV) in six nonresponder patients during three unsuccessful treatments. These patients were treated successively with IFN-alpha2a (IFN-alpha) at a posology of 3.10(6) units (MIU) three times a week, 10 MIU three times a week, and a combination of IFN-alpha (3 MIU) plus ribavirin (1,000 mg/day). However, only two chronically infected patients could be included in the study due to the persistence of HCV RNA during the three successive treatments. The viral diversity was analysed by cloning and sequencing the HVR-1 region. The treatment of the two nonresponder patients was associated with the persistence of a wide diversity in the viral population and with the emergence of new or minor variants. Under the influence of standard doses of IFN-alpha, a rearrangement of the quasispecies present was observed at this time point. No significant change in viral load or in the complexity of the quasispecies was observed. A second treatment with a high dose of IFN-alpha induced a significant decrease in the associated viral load and, in one case, resulted in a radical change of the viral diversity. Administration of a combination of IFN-alpha and ribavirin did not affect the evolution of the variants but was followed by the emergence of various multiple variants. These results reinforce the hypothesis of the presence of preexisting quasispecies best adapted to the host environment, and therefore resistant to any current therapy.
Hepatitis C virus pathogenesis and cycle are difficult to study because of the lack of culture system able to replicate efficiently the virus. Furthermore such a system will permit screen new antiviral drugs. Studies were realized to select cell culture system able to allow hepatitis C virus replication. Primary cell cultures and cell lines were used to performed HCV culture. Most of these works used lymphocyte and hepatocyte primary cultures or cell lines because of HCV tropism in these cells in vivo. Animals and arthropods cell lines were used as well for their capacity to bind and replicate HCV. The aim of this review is to present the different cell systems used to replicate HCV in culture and the results obtained.
Chronic stress is known to induce immunological disorders. In the present study we examined the consequences of chronic restraint stress on the immune response to tetanus toxin in mice. We investigated the repartition of subsets of lymphoid cells in blood and spleen, the functional ability of lymphocytes to proliferate and to produce cytokines, and antibody titres against tetanus toxin following stress. We report discordance of the stimulation index of lymphocytes in the restraint group: the proliferating rate severely decreased following stimulation with a relevant antigen, whereas it increased with mitogen. Thus, we report a decrease in cytokine production with relevant antigen (interferon-gamma and interleukin-10), without a T helper type 1 and 2 secretion imbalance. Moreover, we observed an alteration in the humoral response, including a delay in isotype maturation and an immunoglobulin G1/G2a imbalance.
We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.
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2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidation of hyaluronan was studied at pH 10.2 and temperature of 0 degrees C with NaOCl as the primary oxidant. As with other polysaccharides, a high selectivity of oxidation was observed. The degradation of the polymer was essentially caused by the oxidation process. The primary oxidant and the pH of the reaction mixture did not alter the molecular weight of hyaluronan during oxidation. The kinetics of the oxidation process was investigated at different concentrations of reactants and the inorganic salts, NaBr, NaCl, and Na2SO4. An increase in the salt concentration in the mixture causes a major decrease in the rate of the oxidation, and this decrease is independent of the nature of the salt.
The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of specific viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein-Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective influence of these molecules on the expression of various viral target genes varied greatly. In replication-defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that influence all stages of viral production.
A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.
Hodgkin's disease is commonly associated with EBV latent infection. The incidence of EBV reactivation (active infection or EBV infection with replicative cycle) was evaluated in a series of 30 patients with untreated Hodgkin's disease (except for one case with chronic lymphocytic leukemia) by quantitation of EBV DNA and titration of anti-ZEBRA antibodies in serum samples. DNA was detected in serum (>2.5 x 10(2) genomes/ml) in 15 of 30 patients and was more frequent in Hodgkin's disease with EBV-positive Reed-Sternberg cells (10/12) than in EBV-negative cases (5/18), (P< 0.01). Of interest was the demonstration that viremia correlated well with increased titers of anti-ZEBRA IgG and/or standard serological profiles of EBV reactivation (12/15), (P < 0.05). However the lack of EBV replicative cycle in Reed-Sternberg cells (negative for ZEBRA antigen and early antigen BHLF1) suggests that the viral replication occurs in a nonneoplastic cell compartment rather than in tumor cells. The measurement of EBV DNA loads and the titration of anti-ZEBRA antibodies shed new lights on the link between activation of EBV replication and Hodgkin's disease: these serological markers together with the determination of the EBV status of the tumor suggest that replication of the viral genome occurs with a decreased efficiency of the immune system, thus allowing progression of the tumor.
In the present study we report that the appearance of oligo-monoclonal immunoglobulins (oligoM-Igs) in the sera of transplanted individuals is concurrent with the detection of coincident active CMV infection and EBV replication. Eighty-four renal allograft patients were monitored with respect to CMV isolation, to CMV conventional serology and humoral response against the EBV trans-activator ZEBRA (an immediate-early antigen also called BZLF1). Titration of anti-ZEBRA antibodies (IgG and IgM) and amount of EBV DNA in serum were evaluated. Using the combination of four techniques (agarose gel electrophoresis, analytical isoelectric focusing, high resolution immunoelectrophoresis, immunofixation electrophoresis), oligoM-Igs were found in 25% of patients after allografting and significantly associated with rejection episodes (P < 0.001). Twenty out of 23 (86%) concurrent CMV/EBV infections were associated with serum oligoM-Igs (P < 0.001). One can thus reasonably assume that a sustained EBV replication following iatrogenic immunosuppression can promote the immunoglobulin heavy chain expression in EBV-infected B lymphocytes. The proliferation of immunoglobulin-secreting clones might occur after active CMV infection, through a transient over-immunosuppression or via immune subversion.
Epstein-Barr virus (EBV) is distributed widely throughout the world. Apart from a association with two geographically-restricted malignancies (Burkitt's lymphoma and nasopharyngeal carcinoma), EBV is thought to be implicated in the etiology of B-cell lymphoma in immunocompromised individuals. In these patients, monitoring the viral load in serum can provide useful information on the timing of the instigation of antiviral therapy, i.e. as soon as a rise is detected. PCR technology, owing to its high sensitivity, is used frequently in such situations. In order to gain further insight into the nature of the peripheral blood cells carrying the viral genome on a cell-by-cell basis, an in situ amplification technique was developed as a model using two cell lines growing in suspension, with the aim of distinguishing between EBV-positive and EBV-negative cells. Preliminary experiments were undertaken subsequently on clinical samples from patients with infectious mononucleosis and patients with lymphoma indicating that this technique might be useful clinically.